Publications

• 13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase.

  Hanes J.W., Keresztes I., Begley T.P.

  The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxa ... >> More

  The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate. << Less

  Nat. Chem. Biol. 4:425-430(2008) [PubMed] [EuropePMC]

• Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis.

  Wallner S., Neuwirth M., Flicker K., Tews I., Macheroux P.

  Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heterom ... >> More

  Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heteromeric PLP synthase, consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. l-Glutamine hydrolysis by Pdx2 supplies ammonia to Pdx1 for incorporation into PLP. Autonomous glutaminase Pdx2 is inactive; however, interaction with Pdx1 leads to enzymatic activity. Oxyanion hole formation in the active site of Pdx2 is required for substrate binding and was suggested as the prime event of enzyme activation. Here, we dissect interactions required for complex formation from interactions required for catalytic activation of the glutaminase. The three-dimensional structural analysis suggested a number of invariant residues that regulate complex formation and enzyme activation. We have replaced several of these invariant residues by site-directed mutagenesis in an effort to understand their function. In addition to the biochemical characterization of enzyme activity, the generated protein variants were studied by isothermal calorimetry to investigate their role in complex formation. The assembled data describe a multistep activation mechanism. Residues of helix alphaN of Pdx1 are essential for formation of the Pdx1-Pdx2 complex and also stabilize the oxyanion hole. Thus, these interactions describe the encounter complex. On the other hand, residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 contribute to interface formation and are required for the organization of the catalytic center; thus, these interactions describe the Michaelis complex. However, the main players for formation of the Michaelis complex reside on Pdx2, as replacement of residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 leads to reduction but not complete inactivation of the glutaminase. << Less

  Biochemistry 48:1928-1935(2009) [PubMed] [EuropePMC]

• Reconstitution and biochemical characterization of a new pyridoxal-5'-phosphate biosynthetic pathway.

  Burns K.E., Xiang Y., Kinsland C.L., McLafferty F.W., Begley T.P.

  The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.

  J. Am. Chem. Soc. 127:3682-3683(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of a bacterial pyridoxal 5'-phosphate synthase complex.

  Strohmeier M., Raschle T., Mazurkiewicz J., Rippe K., Sinning I., Fitzpatrick T.B., Tews I.

  Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently ... >> More

  Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel. << Less

  Proc. Natl. Acad. Sci. U.S.A. 103:19284-19289(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Reaction mechanism of pyridoxal 5'-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate.

  Raschle T., Arigoni D., Brunisholz R., Rechsteiner H., Amrhein N., Fitzpatrick T.B.

  Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered an ... >> More

  Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5'-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609-14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism. << Less

  J. Biol. Chem. 282:6098-6105(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Trapping of a chromophoric intermediate in the Pdx1-catalyzed biosynthesis of pyridoxal 5'-phosphate.

  Hanes J.W., Keresztes I., Begley T.P.

  Angew. Chem. Int. Ed. Engl. 47:2102-2105(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis.

  Raschle T., Amrhein N., Fitzpatrick T.B.

  Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restrict ... >> More

  Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex. << Less

  J. Biol. Chem. 280:32291-32300(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanistic studies on pyridoxal phosphate synthase: the reaction pathway leading to a chromophoric intermediate.

  Hanes J.W., Burns K.E., Hilmey D.G., Chatterjee A., Dorrestein P.C., Begley T.P.

  Two routes for the de novo biosynthesis of pyridoxal-5'-phosphate (PLP) have been discovered and reconstituted in vitro. The most common pathway that organisms use is dependent upon the activity of just two enzymes, known as Pdx1 (YaaD) and Pdx2 (YaaE) in bacteria. Pdx2 has been shown to have glut ... >> More

  Two routes for the de novo biosynthesis of pyridoxal-5'-phosphate (PLP) have been discovered and reconstituted in vitro. The most common pathway that organisms use is dependent upon the activity of just two enzymes, known as Pdx1 (YaaD) and Pdx2 (YaaE) in bacteria. Pdx2 has been shown to have glutaminase activity and most likely channels ammonia to the active site of the PLP synthase subunit, Pdx1, where ribose-5-phosphate (R5P), glyceraldehyde-3-phosphate (G3P), and ammonia are condensed in a complex series of reactions. In this report we investigated the early steps in the mechanism of PLP formation. Under pre-steady-state conditions, a chromophoric intermediate (I320) is observed that accumulates upon addition of only two of the substrates, R5P and glutamine. The intermediate is covalently bound to the protein. We synthesized C5 monodeuterio (pro-R, pro-S) and dideuterio R5P and showed that there is a primary kinetic isotope effect on the formation of this intermediate using the pro-R but not the pro-S labeled isomer. Furthermore, it was shown that the phosphate unit of R5P is eliminated rather than hydrolyzed in route to intermediate formation and also that there is an observed C5-deuterium kinetic isotope effect on this elimination step. Interestingly, it was observed that the formation of the intermediate could be triggered in the absence of Pdx2 by the addition of high concentrations of NH4Cl to a preincubated solution of Pdx1 and R5P. The formation of I320 was also monitored using high-resolution electrospray ionization Fourier transform mass spectrometry and revealed a species of mass 34,304 Da (Pdx1 + 95 Da). These results allow us to narrow the mechanistic possibilities for the complex series of reactions involved in PLP formation. << Less

  J. Am. Chem. Soc. 130:3043-3052(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.